1. Technical Field
The present invention is directed to reagents and methods for performing an immunoassay, particularly a fluorescence polarization immunoassay (FPIA), to determine the presence and/or amount of dextropropoxyphene and/or the principal metabolite of dextropropoxyphene (namely, nordextropropoxyphene) in samples, particularly aqueous, fluid biological samples such as urine, blood serum or blood plasma, and to an immunoassay based on the reagents. More particularly the invention is directed to new haptens, immunogens prepared from the haptens, antibodies raised against the haptens and immunoassays which utilize reagents and methods of the invention.
2. Background
Dextropropoxyphene is a narcotic analgesic which has found wide therapeutic use. Unfortunately, however, it has also become a drug of abuse. Dextropropoxyphene is also known by the following chemical names: [S-(R*,S*)]-alpha-[2-(Dimethylamino)-1-methylethyl]-alpha-phenylbenzeneeth anol propanoate (ester); alpha-d-4-Dimethylamino-3-methyl- 1,2-diphenyl-2-butanol propionate; (+)- 1,2-Diphenyl-2-propionoxy-3-methyl-4-dimethylaminobutane; (+)-4-Dimethylamino- 1,2-diphenyl-3-methyl-2-propionyloxybutane; alpha-d-4-Dimethylamino-3-methyl- 1,2-diphenylbutan=2-ol propionate; and d-propoxyphene. Dextropropoxyphene corresponds to the following structural formula: ##STR1##
As reported by R. J. Flanagan et al in "Measurement of Dextropropoxyphene and Nordextropropoxyphene in Biological Fluids", Human Toxicol. (1984), 3,103S-114S, various methods have been explored for detecting, identifying and measuring the amount of dextropropoxyphene and its principal plasma metabolite, nordextropropoxyphene. Examples of such methods include thin-layer chromatography (TLC) and gas-liquid chromatography (GLC) of solvent or solid-phase extracts of urine or gastric contents, as well as a homogeneous enzyme immunoassay method.
U.S. Pat. No. 4,025,501 is directed to compounds for conjugation to antigens for the production of antibodies which recognize (d,l)-propoxyphene and its metabolites in immunoassays. The compounds to which this patent is directed are prepared by reacting 1,2-diphenyl-3-methyl-4-dimethylamino-2-butanol with a dibasic acid to form a half-acid ester, the acid group of which is disclosed as being activated for conjugation to amino groups of proteins or polypeptides.
However, presently existing assays for propoxyphene tend to suffer from disadvantageously high cross-reactivities for various structurally similar compounds. The present invention has a number of objects. They include, for example, providing new haptens, immunogens prepared from the haptens, and antibodies raised in response to the immunogens, suitable for use in immunoassays which are highly discriminating for dextropropoxyphene and its principal metabolite, nordextropropoxyphene, in which immunoassays cross-reactivity for interfering compounds such as methadone and chlorphenoxamine, among others, is minimized or substantially eliminated.
Additionally, presently existing assays for propoxyphene involve the use of racemic mixtures of molecules for preparation of immunogens to raise antibodies against propoxyphene. However, propoxyphene can exist in four different isomeric forms because of the presence of two optically active centers within the molecule. Thus, utilization of such racemic mixtures for the preparation of immunogens to raise antibodies is believed to result in the production of at least four types of antibodies of which only about 25% will detect the optical form of propoxyphene that is relevant to the assay. The form of propoxyphene which is available on the market is an optically active form of the drug. Moreover, of the four isomeric forms of propoxyphene, there is only one form which appears to have any substantial efficacy in man, namely dextropropoxyphene. Accordingly, particularly for quantitative assays for that form of propoxyphene having efficacy in man, and/or for its most important metabolite, it would be desirable to provide antibodies, a larger proportion of which are reactive to the effective form of the drug than to ineffective forms of the drug.
The present invention is related to immunoassays, particularly competitive immunoassays, involving reagents and techniques particularly suitable for determining the presence and/or amount of dextropropoxyphene and/or nordextropropoxyphene in biological fluids. The present invention can provide, among others, an advantage of allowing for an advantageously effective determination of the amount of dextropropoxyphene and/or its principal metabolite, nordextropropoxyphene, with minimization of interference from other related compounds. The present invention is in part based on new, substantially optically pure haptens, which can be utilized in the preparation of immunogens and/or tracers suitable for use in immunoassays.